WT and mutant CA proteins were expressed from pET3a-CA in E. coli and purified as described52 . The quadruple CA mutant for hexamer formation was expressed from pET15b-His-CAA14C/E45C/W184A/M185A and purified as described53 . For preparation of all GST fused proteins the coding sequences of indicated protein constructs were engineered in the pEX plasmid. The recombinant proteins were expressed in E. coli and purified through two column chromatography steps using 5 ml GSTrap 4B and HiTrap Q High Performance 5 ml columns (GE Healthcare). Point-mutations and amino acid deletions in the protein of interest were introduced via site-directed mutagenesis (Quickchange II XL site-directed mutagenesis kit, Agilent). Recombinant Sec23A/Sec24C and Sec23A/Sec24D proteins24 (link) were a gift from Robert Lesch, University of California Berkeley. Protein concentrations were analyzed by using Enspire manager (3.10.3005.1440).
Hitrap q high performance 5 ml columns
Manufactured by GE Healthcare
The HiTrap Q High Performance 5 ml columns are a pre-packed anion exchange chromatography column designed for rapid and convenient protein purification. The columns provide high-resolution separation and can be used for a variety of biomolecules, including proteins, peptides, and nucleic acids.
Automatically generated - may contain errors
2 protocols using hitrap q high performance 5 ml columns
1
Expression and Purification of Recombinant Proteins
2
Expression and Purification of Recombinant Proteins
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WT and mutant CA proteins were expressed from pET3a-CA in E. coli and purified as described52 . The quadruple CA mutant for hexamer formation was expressed from pET15b-His-CAA14C/E45C/W184A/M185A and purified as described53 . For preparation of all GST fused proteins the coding sequences of indicated protein constructs were engineered in the pEX plasmid. The recombinant proteins were expressed in E. coli and purified through two column chromatography steps using 5 ml GSTrap 4B and HiTrap Q High Performance 5 ml columns (GE Healthcare). Point-mutations and amino acid deletions in the protein of interest were introduced via site-directed mutagenesis (Quickchange II XL site-directed mutagenesis kit, Agilent). Recombinant Sec23A/Sec24C and Sec23A/Sec24D proteins24 (link) were a gift from Robert Lesch, University of California Berkeley. Protein concentrations were analyzed by using Enspire manager (3.10.3005.1440).
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